Cells of both invertebrate and vertebrate embryos that give rise to neurons must correctly receive and interpret positional information throughout development so that terminally differentiated neurons are precisely determined. The mis- interpretation of positional cues can lead to severe developmental abnormalities and in the most extreme case death. Ventral nervous system defective (vnd), an NK-2 type homeobox gene, specifies the identity of midline proximal ventral neuroectodermal cells and neuroblasts in Drosophila that give rise to motor neuron and interneurons. Since the vertebrate homologues of this gene, Nkx2.2 and Nkx 2.1, play parallel roles in vertebrate neuronal patterning, this suggests both regulatory and functional conservation. The overall goal of this proposal is to understand how the Drosophila vnd gene integrates positional information so that it fits into the hierarchical network involved in CNS dorsal-ventral specification. Specifically, we will localize the enhancers responsible for vnd- specific expression by dissecting the 5' region of vnd in transgenic embryos. In addition we will identify conserved sequence islands in the vnd regulatory domain from two related Drosophila species. We will perform DNA binding studies with those regulators that bind vnd enhancers. We will confirm the functional significance of the DNA binding sites by mutating these regions and testing the activity of mutated enhancers in a transgenic embryo reporter assay system. We will also address the role of the NK-2 box, a conserved stretch of 18 amino acids, downstream of the homeobox in Vnd's role as a transcription factor. The capacity of mutated Vnd protein, lacking the NK-2 box, to drive reporter expression will be assayed in both tissue culture and transgenic embryos. We recently showed that over- expression of wild-type Vnd leads to a transformation in neuronal precursor identity. Transgenic embryos that over-express mutant Vnd, lacking the NK-2 box, will be assayed for alteration in neural precursor identity. Finally, we will illucidate the relationship between the epidermal growth factor (EGF) pathway and Vnd. EGF patterns the developing CNS along the dorsal- ventral axis by inducing the phosphorylation of largely unidentified target proteins. Since expression of Vnd is altered when the EGF receptor is missing or the EGF ligand, Spitz is over-expressed, Vnd is likely regulated by phosphorylation. The amino acids that are phosphorylated in Vnd will be identified by acid hydrolysis and peptide mapping. Following their mutation, the role of phosphorylation in Vnd's function will be analyzed by examining neuronal marker expression in gain-of-function embryos that over-express wild-type and mutant Vnd. These studies address the structure and function of vnd, a critical regulator of early neuronal development, and may serve as a prototype for vertebrate vnd-like genes.